NO300244B1 - Method for Determining an Analyte Using a Heavily Soluble Salt of a Heteropoly Acid and Agent for Use in the Determination - Google Patents

Abstract

A sparingly soluble salt of a heteropoly acid is used for the determination of an analyte which is an aromatic amine rich in electrons or together with another substance leads to such an amine. The analyte is determined by means of the heteropoly blue color formed.

Description

The present invention relates to the use of a sparingly soluble salt of a heteropolyacid for the determination of an analyte. The invention also relates to a method for determining an analyte by means of the formation of heteropoly blue and an agent suitable for this purpose. The invention also relates to the use of a sparingly soluble salt of a heteropoly acid for the production of a means for determining an analyte through the formation of heteropoly blue.

Heteropolyacids are inorganic polyacids with at least two different central atoms. They consist of polybasic oxygen acids of a metal, such as molybdenum, tungsten, vanadium, and a non-metal or metal, such as phosphorus, silicon, arsenic, iodine, iron, manganese, cobalt, as partial mixed anhydrides. Examples are molybdenum phosphoric acid or tungsten phosphoric acid. Heteropolyacids are water soluble, but they are however

stable in acidic solution.

Heteropolyacids of molybdenum and tungsten have been known for a long time. These reagents are used analytically for the determination of phosphate and also for the detection of arsenate or silicate through the formation of the corresponding heteropolyacids with molybdate or tungstate and subsequent reduction of the heteropolyacids to a blue dye, the so-called heteropolyblue. The color is due to light absorption due to electron transfer between penta- or hexavalent molybdenum or tungsten. Heteropolyblue from heteropolyacids is a dye with high extinction, which, depending on the wavelength, exhibits a very broad absorption maximum. This is typical of "charge-transfer" absorption bands.

Heteropolyacids for the detection of reducing compounds by means of the formation of heteropoly blue are known. In "Spot Tests in Organic Analysis" by F. Feigl, Elsevier Publishing Company, 5th edition, 1956, pp. 128 and 129, it is described that 12-molybdenum phosphoric acid is reduced to molybdenum blue by means of several reducing substances. Because this detection reaction is non-specific, in order to achieve a good selectivity it is recommended to alkalize the probe to be examined, followed by extraction with ether. The ether extract obtained contains above all aromatic nitrogen bases and ether-soluble neutral substances. Acidic substances remain dissolved in the aqueous phase.

From P.B. Issopoulos, Pharm. Acta Heiv. 64, 82 (1989), it is known that certain drugs containing an o-hydroxyquinone structure, such as e.g. methyldopa, in sulfuric acid solution has a reducing effect on molybdenum phosphoric acid and leads to the formation of molybdenum blue.

In M.L. Matheke et al., Clin. Chem. 33, 2109-2110

(1987), describes the use of phosphortungstic acid for the detection of uric acid. The reductive formation of tungsten blue serves as an indicator of the presence of uric acid. The detection reaction is disturbed by reducing drugs present. From B. Klein et al., Clin. Chem. 12, 816-823 (1966), a determination of glucose in serum or plasma is known which is based on the following reaction sequence: Glucose is oxidized with the help of potassium hexacyanoferrate (III), whereby the formed potassium hexacyanoferrate (II) has a reducing effect on phosphormolybdic acid and leads to the formation of molybdenum blue. Because serum and plasma can contain different amounts of uric acid, drugs or other reducing substances, such as e.g. bilirubin and glutathione, by this method their disruptive influence is programmed in advance. The shortcoming of all the hitherto known analytical methods which make use of the reductive formation of heteropolyblue from heteropolyacids is due above all to the non-specificity of the detection reaction in question. Very many reducing compounds can be disruptive because they also lead to the formation of heteropoly blue. Furthermore, heteropolyacids are only stable under acidic conditions. This narrows the area of application to a very high degree. In particular, the connection of the formation of heteropoly blue with enzymatic reaction steps to specific detection and specific determination of compounds is not known so far. Precisely for the detection of components of body fluids such as blood, serum, plasma, urine etc., however, enzymatic determination methods are often necessary. The aim of the present invention was to use the formation of heteropoly blue as a detection and determination reaction of compounds, especially of compounds in body fluids, and especially in combination with enzymatic reaction steps. Furthermore, impurities with a reducing effect should not be disruptive, and the detection and determination reactions should proceed at the required pH values for enzymatic reactions. The detection and determination reactions should also be able to be carried out quickly. It has been found in the present invention that a sparingly soluble salt of a heteropolyacid can advantageously be used for the determination of an analyte, especially when the analyte is an electron-rich, aromatic amine or when the analyte together with a further compound leads to such an amine. The method according to the present invention for determining an analyte by means of the formation of heteropoly blue is characterized by the analyte being an electron-rich, aromatic amine or that the analyte is reacted with a compound, or an enzyme, which leads to the formation of an aromatic, electron-rich amine which is brought into contact with a sparingly soluble salt of a heteropolyacid. The aim of the present invention is achieved by using a sparingly soluble salt of a heteropoly acid to produce a means for determining an analyte through the formation of heteropoly blue. According to the invention, a means for determining an analyte is produced through the formation of heteropoly blue, which is characterized by the fact that it contains a compound which, together with the analyte, leads to the formation of an electron-rich, aromatic amine and, moreover, a sparingly soluble salt of a heteropoly acid or substances formed from contact with such a salt. If the agent according to the present invention is to be used for the direct determination of an electron-rich, aromatic amine, it is sufficient that the agent contains a sparingly soluble salt of a heteropolyacid or substances that are formed by contact with such a salt in a liquid medium or bound to a carrier . According to the present invention, a sparingly soluble salt of a heteropolyacid can be used for the determination of an analyte. A sparingly soluble salt of a heteropolyacid according to the present invention shall above all be understood as a salt of a heteropolyacid which is insoluble or very sparingly soluble in water or aqueous media, such as buffers or body fluids, such as e.g. blood, plasma, serum, urine or saliva, and which also remains under the test and color formation conditions. In particular, the salt of a heteropolyacid is so sparingly soluble that the maximum amount that is soluble in a liquid probe is not alone sufficient for the determination of the analyte contained therein. It has surprisingly been shown that such a sparingly soluble salt of a heteropolyacid is not only stable in acids, but also in neutral and basic pH ranges, especially up to a pH of approximately 10. pH ranges can thus be used where most enzymes are active and thereby lead to the formation of heteropoly blue in combination with enzymatic reactions. Above all, in an undissolved state, a sparingly soluble salt of a heteropolyacid according to the invention is stable even at elevated temperatures. Despite the heavy solubility, it has been shown that an analyte using a salt of a heteropoly acid according to the invention can be detected and determined very quickly, i.e. within a few seconds to a few minutes, through the formation of heteropoly blue. It is thereby possible to achieve a selective determination without interference from other intrinsically reducing substances. This allows a sensitive measurement method which, especially due to the broad absorption maximum for the formed heteropoly blue, which extends from approximately 550 to higher than 100 nm, does not make any special demands on measuring equipment, and which can also be easily monitored visually. Useful salts of heteropolyacids according to the present invention are salts of heteropolyacids of molybdenum, molybdenum and tungsten, vanadium and molybdenum or vanadium and molybdenum and tungsten, with phosphorus, arsenic, silicon or germanium as heteroatoms. Particularly preferred are heteropolyacids of molybdenum with phosphorus or arsenic. Phosphorus is particularly preferred as the non-metal atom. Molybdenum is preferably preferred as the metal atom. Particularly suitable are poorly soluble salts of 12-molybdenum phosphoric acid, 18-molybdenum diphosphoric acid, 12-molybdenum arsenic acid, 18-molybdenum diarsenic acid, 11-molybdenum-1-vanadium phosphoric acid, 10-molybdenum-2-vanadium phosphoric acid and 9-molybdenum-3-vanadium phosphoric acid, whereby 18-molybdenum diphosphoric acid is particularly preferred due to the high color yield of wood

reduction to molybdenum blue.

Sparingly soluble salts of heteropolyacids are those whose cations are larger than ammonium, NH4<+.> Preferred cations can be prepared by means of the general formula I

in which

R1, R<2>, R3 and R<4> are the same or different and denote an alkyl radical, aryl radical or aralkyl radical,

or hydrogen, when all residues are not equal, or

in which two residues together form an alkylene residue, and X denotes a phosphorus atom or nitrogen atom.

In alkyl residues and aralkyl residues, alkyl denotes a straight-chain or branched hydrocarbon residue with 1-22 carbon atoms, preferably 1-6 carbon atoms. Aryl in aryl residues or aralkyl residues denotes an aromatic residue with 6-10 carbon atoms. Particularly preferred is phenyl or naphthyl.

As an aralkyl residue, the benzyl group is particularly preferred.

An alkylene residue is a saturated or unsaturated hydrocarbon chain with 4-6, preferably 4 or 5, carbon atoms, which is bonded to X at both ends. Cations of general formula I can contain two such alkylene residues. Preferably, however, only one alkylene residue is present.

In the general formula I, X preferably denotes a nitrogen atom. Likewise, preferred cations in sparingly soluble salts of heteropolyacids can also be from the group of nitrogen heteroaromatics containing a quaternary N atom. For example, pyridine or quinoline can be mentioned, with an alkyl residue or aralkyl residue attached to the nitrogen atom, whereby the definition of the residues R<1>, R2, R3 and R<4> has the same meaning as indicated under formula I above. A sparingly soluble salt of a heteropolyacid according to the present invention can, for example, be obtained by reacting a heteropolyacid with a corresponding basic compound. For this, as a rule, at least one compound is used in the form of a solution, preferably an aqueous solution. However, both salt components in solid form can also be added to a liquid, especially an aqueous liquid, or vice versa.

An analyte is a compound to be determined. The present invention has proven particularly suitable for compounds that are dissolved in a liquid, especially an aqueous liquid. Particularly advantageously, a sparingly soluble salt of a heteropolyacid can be used for the determination of compounds in body fluids, such as e.g. blood, plasma, serum, urine or saliva. As possible analytes in this connection, e.g. mention is made of glucose, cholesterol, lactate, NADH or ethanol. According to the present invention, in principle all those compounds can be determined which, together with one or more other compounds, can be converted into such analytes, or which themselves are such analytes, and which, with regard to their redox potentials and their kinetics, are in a state that they can reduce a poorly soluble salt of a heteropoly acid to heteropoly blue within a few seconds to a few minutes, preferably less than 3 min. It has surprisingly been shown that electron-rich, aromatic amines have this property. Above all, it is surprising that a selective determination without interference by other intrinsically reducing compounds is possible.

An electron-rich, aromatic amine is to be understood as a compound that is more electron-rich than aniline and thereby represents a stronger reducing agent. Electron-rich aromatic amines have a redox potential of less than 0.6 V, preferably less than 0.45 V, against a normal hydrogen electrode. However, the size of the redox potential alone is not decisive. It is also important that the corresponding compound is capable of reducing a sparingly soluble salt of a heteropoly acid to heteropoly blue, i.e. within less than approximately 3 min. Such applicable compounds are, for example, all aniline derivatives, to which one or more +1- or/and +M-substituents are attached, such as hydroxy-, alkyl-, alkoxy-, aryloxy-, alkylthio-, arylthio-, amino-, monoalkylamino- and dialkylamino residues.

Alkyl, alkoxy, alkylthio, monoalkylamino and dialkylamino residues are residues in which the alkyl group represents a hydrocarbon residue with 1-6 carbon atoms, which in turn may be substituted with a hydroxy group, possibly an amino group which is mono- or poly-substituted with alkyl with 1-6 carbon atoms, H2P03, HS03 or HC02. The acid residues H2P03, HS03 and HC02 can be present as such residues or in salt form such as ammonium, alkali or alkaline earth salts.

Aryloxyacid residues and arylthio residues are residues with 6-

10 carbon atoms, where phenoxy acid residues and phenylthio residues are particularly preferred.

Aniline derivatives are also understood to mean compounds with an unsubstituted amino group or an amino group that is mono- or poly-substituted with alkyl, bound to an aromatic ring system that is ortho-connected with one or more aromatic and/or alicyclic rings. Aromatic rings can be both carbonaromatic systems and heteroaromatics. Examples are ortho-connected benzene or naphthalene rings or an ortho-connected pyridine ring.

Alicyclic rings denote saturated or unsaturated cycloaliphates with 5-7 carbon atoms, preferably 5 or 6 carbon atoms.

Possible alkyl substituents on the amino group can be hydrocarbon residues with 1-6 carbon atoms, which in turn can be substituted with a hydroxy group, an amino group which is optionally mono- or poly-substituted with alkyl with 1-

6 carbon atoms, H2P03, HS03 and HC02. The acid residues H2P03, HS03 and HC02 can be present as such or in salt form such as ammonium, alkali or alkaline earth salts. Particularly suitable as electron-rich, aromatic amines are compounds of general formula II

in which

R<5> denotes hydroxy or amino, where the amino group is optionally substituted with 1 or 2 alkyl residues and the alkyl residue in turn is optionally substituted with a hydroxy group, an amino group which is optionally mono- or poly-substituted with alkyl, H2PO3, HS03 or HC02,

R6 and R7 are either the same or different and denote hydrogen or alkyl, where the alkyl residue is optionally substituted with a hydroxy group, an amino group which is optionally mono- or poly-substituted with alkyl,

H2P03, HS03 or HC02, and

R<8>, R9, R<10> and R<11> are either the same or different and denote hydrogen, alkyl, alkoxy, alkylthio, aryloxy, arylthio, halogen, carboxy, carboxyalkyl or alkoxycarbonyl.

Alkyl residues and "alkyl" in alkylthio, carboxyalkyl and alkoxycarbonyl residues are hydrocarbon residues with 1-6 carbon atoms. Particularly preferred are residues with 1-3 carbon atoms.

Alkockic acid residues are likewise hydrocarbon residues with 1-

6 carbon atoms. Particularly preferred are residues with 1-3 carbon atoms. Aryloxy and arylthio residues are residues with 6-10 carbon atoms, whereby phenoxy and phenylthio residues are particularly preferred. Halogen denotes fluorine, chlorine, bromine or iodine. Chlorine and bromine are preferred halogen substituents.

The acid residues H2P03, HS03 or HC02 can be present as such or in salt form as ammonium, alkali or alkaline earth salts.

Ammonium salts are salts containing the ammonium ion, NH4<+>, or such salts containing ammonium cations which are mono- or poly-substituted with alkyl, aryl or aralkyl residues. Alkyl in alkyl and aralkyl residues denotes a hydrocarbon residue with 1-6 carbon atoms. Aryl in aryl and aralkyl residues is an aromatic ring system with 6-10 carbon atoms, where phenyl is preferred. A preferred aralkyl radical is benzyl.

Alkali salts are preferably salts of lithium, sodium or potassium. Alkaline earth salts are preferably salts of magnesium or calcium.

Electron-rich aromatic amines can be immediately determined using a sparingly soluble salt of a heteropoly acid through the formation of heteropoly blue. However, it is also possible to determine compounds which are stoichiometrically converted into an electron-rich, aromatic amine by means of a chemical or enzymatic reaction with a further compound. For example, such compounds can be used as reaction partners whose basic skeleton already contains an electron-rich, aromatic amine which can be released by means of a chemical or enzymatic reaction with the analyte to be determined. This should preferably be understood as such compounds which form an electron-rich, aromatic amine through hydrolysis. Examples include amides, esters or glycosides of electron-rich, aromatic amines. Analytes that can be determined in this way are preferably enzymes that catalyze the conversion of the corresponding substrates into electron-rich, aromatic amines, in particular hydrolase that cleaves amide bonds and/or peptide bonds, enzymes that cleave ester bonds such as carboxylic acid bonds, phosphoric acid bonds or sulfuric acid bonds, and enzymes which cleave glycosidic bonds, also transferases which catalyze the transfer of groups, such as e.g. Y-glutamyl transferase.

For example, determination of compounds that can be oxidized enzymatically can also be carried out, and furthermore compounds that form electron-rich, aromatic amines through reduction can be introduced as electron acceptors. Above all, for substances in body fluids, such as blood, plasma, serum, urine or saliva, a large number of oxidoreductases are known which recognize specifically determined compounds and which oxidize these compounds in the presence of a functioning electron acceptor.

Examples are flavin-dependent oxidases such as L- and D-amino acid oxidase, cholesterol oxidase, glucose oxidase, glycerol-3-phosphate oxidase, lactate oxidase or pyruvate oxidase and non-NAD(P)-dependent dehydrogenases such as pyrrolo- quinoline-quinone-dependent glucose dehydrogenase or also diaphorase (NADH: dye oxidoreductase).

In the case of oxidoreductases, especially oxidases and non-NAD(P)-dependent dehydrogenases, electron acceptors can be added for enzymatic oxidation that is reduced to an electron-rich aromatic amine through the enzymatic oxidation of the enzyme substrate. In this way, compounds that are oxidized enzymatically can be detected and determined by bringing the formed electron-rich aromatic amine into contact with a sparingly soluble salt of a heteropolyacid that gives rise to heteropolyblue.

As electron acceptors according to the present invention, special mention must be made in this connection of aromatic nitroso compounds, oximes and hydroxylamines. Particularly preferred are aromatic nitroso compounds and oximes. Particularly preferred are such nitroso compounds and oximes as are described in European patent application EP-A-0,354,441.

To carry out the method according to the present invention for determining an analyte through the formation of heteropoly blue, it is sufficient that the analyte, as described above, is reacted with a compound and converted to an electron-rich, aromatic amine and brought into contact with a sparingly soluble salt of a heteropolyacid. When the electron-rich aromatic amine itself is the analyte to be determined directly, this is brought into contact with a sparingly soluble salt of a heteropolyacid without prior chemical or enzymatic reactions.

As a rule, at least the electron-rich, aromatic amine which acts on the salt of the heteropolyacid is present in dissolved form, preferably in an aqueous solution, for example water, buffer or body fluid. When the analyte to be determined is not itself an electron-rich, aromatic amine, the necessary compound which together with the analyte produces an electron-rich, aromatic amine is also soluble in aqueous liquids. The aqueous solution can be added to the probe before it is contacted with a sparingly soluble salt of a heteropolyacid. However, the solution can also first be brought into contact with the sparingly soluble salt of a heteropolyacid or even finally brought into contact with the probe to be examined. Which order is chosen depends on the individual case and can be decided by the expert on the basis of general professional knowledge or on the basis of a few optimization attempts. The compound necessary to form an electron-rich aromatic amine can be added to an aqueous probe in solid or dissolved form.

The compound which, together with the analyte to be determined, forms an electron-rich, aromatic amine, must be brought into contact with the probe in at least such a large amount that the total amount of analyte can be brought into reaction. Preferably, a 2-10 times excess is added.

The sparingly soluble salt of a heteropolyacid can, in the form of a solid, be brought into contact with the probe to be examined. However, the salt can also be present in the form of a suspension in a liquid, preferably an aqueous liquid, and in order to carry out the analysis, the suspension is mixed with the probe to be examined. In the presence of the analyte to be determined, heteropoly blue, which is poorly soluble in water and recognizable due to its intense color, is formed. For quantitative determination, it is possible to separate the sparingly soluble heteropolyblue as well as unreacted heteropolyacid salt from the liquid, for example by means of centrifugation, followed by dissolution in a suitable solvent such as dimethylsulfoxide and photometric measurement of the concentration of heteropolyblue dye, possibly with reference to standard solutions or standard curves, thereby determining the concentration of the analyte to be detected.

The sparingly soluble salt of a heteropolyacid must be present in such a large amount that the electron-rich, aromatic amine present or formed in the probe leads to the formation of heteropoly blue, which can be put into a quantitative relationship with the amount of the analyte or the electron-rich aromatic amine to be determined. In principle, such a large amount of sparingly soluble salt of a heteropolyacid must therefore be added that even with the highest relevant concentration of the electron-rich, aromatic amine, an increase in the amount of amine will lead to an increase in the amount of heteropoly blue.

A means according to the present invention for determining an analyte through the formation of heteropoly blue contains a compound which, together with the analyte, leads to an electron-rich, aromatic amine and a sparingly soluble salt of a heteropoly acid. Such an agent can, for example, be added in the form of a suspension or a lyophilisate, a powder mixture or pressed into tablets. The components can be present together or separately, whereby each of the components can be processed into its most suitable form. It is also absolutely possible that an agent according to the invention does not contain a ready-made sparingly soluble salt of a heteropolyacid, but only the necessary components for the preparation of such a salt, namely, for example, a heteropolyacid and a cationic or basic compound, both of which are brought into contact with each other only immediately before the determination reaction according to the invention and only then does it form the corresponding salt. It is particularly advantageous when the agent according to the present invention contains a sparingly soluble salt of a heteropolyacid in finely divided form, so that it exhibits a large reactive surface. The agent according to the invention may possibly contain further reagents such as buffers and excipients, such as e.g. wetting agents, stabilizing agents and structure-forming agents. If the analyte to be determined is itself an electron-rich, aromatic amine, the agent according to the invention does not of course need to contain a compound which forms an electron-rich, aromatic amine only after reaction with the analyte.

The determination of an analyte according to the present invention is particularly advantageously carried out using a so-called dry test. Devices which are suitable for carrying out a dry test are known to the person skilled in the art from, for example, EP-A-0.016.387, DE-A-3.247.608, EP-A-0.262.445 or EP-A-O.256.806. The test devices can be described as test carrier materials. For carrying out a test, the necessary reagents are available here in dry form, i.e. not dissolved in a liquid, in or on an absorbent material such as e.g. paper, an open film according to EP-A-0,016,387, glass fiber wool or a porous plastic membrane, or in or on a swellable material such as e.g. a corresponding film of plastic, gelatin or cellulose.

When the probe-containing liquid is applied to the test carrier material or when the test carrier material is immersed in the probe-containing liquid, a liquid environment is formed in the test carrier material, within which the detection reaction takes place. The color formation caused by the reaction can be evaluated visually or photometrically, e.g. reflectance photo-metric.

To produce the agent according to the present invention in carrier-bound form, a suitable carrier material, such as filter paper, cellulose or synthetic fiber wool, is impregnated with the necessary solutions and/or suspensions of reagents in easily volatile solvents, such as e.g. water, methanol, ethanol or acetone, which are used in the manufacture of a test device. This can take place in an impregnation step. It is often appropriate for the impregnation to be carried out in several stages, whereby solutions and/or suspensions are added which possibly contain one part of the components in the finished product. In a first step, for example, a suspension of a sparingly soluble salt of a heteropolyacid can be applied to a carrier material, and in a second step a solution of a compound which, together with the analyte to be determined, gives an electron-rich, aromatic amine, which solution also possibly contain buffer and other additive materials. The carrier material treated in this way is used as such or glued to a rigid substrate, such as a rigid plastic film, in order to be handled more easily.

Instead of a multi-stage impregnation of the same carrier material, the reagents in the agent according to the present invention can also be distributed on different carrier materials, which when carrying out the analytical determination can be brought into contact with each other through a liquid exchange.

For the production of carrier-bound test agents, instead of an impregnation, film-forming components can also be used, i.e. polymers or polymer dispersions, from which solutions can be produced that are so viscous that they can be used to produce films using known production methods, such as coating and roller coating . The reagents and possibly buffer substances and auxiliary materials are incorporated into these solutions. The coating compounds are applied to carrier foils, dried, and the finished films are processed, e.g. to test strips. Figures 1 and 6 show examples of preferred test devices. The other figures 2-5 and 7 show diagrams of the reflection in percentage as a function of time, wavelength or analyte concentration, obtained with test devices according to respectively figure 1 and figure 6. Figure 1: a cross-section through a preferred test device. Figure 2: a diagram showing the reflectance in percent as a function of time in seconds after applying a probe to a test device according to Figure 1 for the probes a)-f) with different glucose concentrations. Figure 3: a diagram showing the reflection in percent as a function of the wavelength in nanometers for the probes a)-f) with different glucose concentrations using a test device according to Figure 1. Figure 4: a diagram showing the reflection in percent as a function of the glucose concentration measured with a test device according to Figure 1. Figure 5: a diagram showing the reflectance in percent as a function of the NADH concentration measured with a test device according to Figure 1. Figure 6: a cross section through a further preferred test device. Figure 7: a diagram showing the reflectance in percent as a function of the ethanol concentration measured with a test device according to Figure 6.

The following examples show some of the possible method variants for determining an analyte through the formation of heteropoly blue. However, these embodiments are not limiting for the present invention. The figures are explained in more detail in the examples.

Example 1

Detection of glucose with 18- molybdenum diphosphate

A test device according to figure 1 is produced. It consists of a 350 µm thick polyester foil as carrier foil (1) with the measurement 2 x 3 cm. In the center of the foil there is an opening with a diameter of 6 mm. Using an adhesive (2), the reagent carrier (6), consisting of a 200 µm thick, transparent polycarbonate foil (5), a first reagent layer (4) and a second reagent layer (3), is fixed over the opening in the carrier foil in such a way that the probe liquid which is applied through this opening first comes into contact with the second reagent layer. The reagent carrier has the dimensions 2 x 1 cm.

Preparation of the reagent carrier (6) is carried out as follows:

First reagent layer:

stirred to a homogeneous mass and applied to the transparent foil (5) in a thickness of 150 µm. The layer is dried for 1 hour at 60 °C.

Second reagent layer:

stirred to a homogeneous mass and applied to the first reagent layer in a thickness of 400 µm. Air bubbles are removed by careful air blowing, and the layer is dried for 1 hour at 60 °C. At time t = 0, human plasma is added with a) 0 mg glucose/dl, b) 47.5 mg glucose/dl, c) 108.7 mg glucose/dl, d) 200.8 mg glucose/dl, e) 392 .3 mg glucose/dl and f) 766 mg glucose/dl on a test device prepared as described above. The percentage remission is measured at 950 nm. It

the first measurement is taken after 8 seconds, and further measurements are taken at 4 second intervals. When plotting the remission (R) in percentage against the time (t) in seconds, a diagram is obtained as shown in figure 2. The color formation and remission reduction are already complete at the first measurement. A stable final value was obtained which can be measured at almost any time after the start.

If samples with a glucose concentration of a)

0 mg/dl, b) 100 mg/dl, c) 240 mg/dl and d) 800 mg/dl are measured at

If samples with a glucose concentration of a)

0 mg/dl, b) 100 mg/dl, c) 240 mg/dl and d) 800 mg/dl are measured at different wavelengths using a test device according to Figure 1, prepared as described above, and if the remission (R) in percentage is plotted against the wavelength (A) in nm, a diagram is obtained as shown in figure 3. As shown in the depicted curves, any wavelength between 550 nm and up to higher than 1100 nm can be used to measure the glucose concentration. Due to the extremely flat spectra, the tolerance to changes in wavelengths used for measurement is very high.

Below is shown a reaction core that shows how glucose is converted into an electron-rich aromatic amine.

Detection of glucose with 18-molybdenum diphosphate (Example 1)

Example 2

Specificity of glucose detection with 18-molybdenum diphosphate

The interfering substances listed in Table 1 are added in the indicated concentrations to human plasma with a) 0 mg glucose/dl (Cgluc = 0) and b) 110 mg glucose/dl (Cgluc <=>

110 mg/dL). If such a human plasma is examined with a test device according to figure 1, prepared and described as in example 1, remission values in percent (% R) are obtained at 660 nm as indicated in table 1.

Regardless of the glucose concentration in human plasma (0 and 110 mg/dl), no significant disturbances were found.

With a test device according to figure 1 which is produced analogously to the device according to example 1 and which is identical to the test device described therein, with the exception that tetra-butylammonium chloride is omitted in the comparative test device, very poorly reproducible, strongly scattered results are obtained due to that the added 18-molybdenum diphosphoric acid decomposes at pH values higher than 5.5. The reducing substances also have a disruptive effect here through further color formation.

Example 3

Test device for the detection of glucose on the basis of 12-mol vbdenophosphate

By replacing 18-molybdenum diphosphoric acid in the recipe according to example 1 with the same amount of 12-molybdenum phosphoric acid, a test device is obtained with weaker color formation in the presence of glucose which is particularly suitable for higher glucose concentrations. When using probes with different but known glucose concentrations (C) and when measuring the corresponding remissions (R) in percentage at 650 nm, the curve according to Figure 4 is obtained. When measuring at 950 nm, an identical curve is obtained.

Similar results are obtained with 12-molybdenum arsenate (V), 18-molybdenum diarsenate (V), 11-molybdenum-1-vanadium phosphate, 10-molybdenum-2-vanadium phosphate and 9-molybdenum-3-vanadium phosphate, all of which can be prepared according to G. Brauer , "Handbuch der praparativen anorganischen Chemie", Enke-Verlag, Stuttgart,

(1954). In the following table 2 are listed the names and formulas of the heteropoly acids as well as the absorption maximum for the corresponding heteropoly blue.

Example 4

Test device for the determination of NAD(P)H

By replacing the glucose oxidase in the recipe according to example 1 with the same amount of diaphorase, a test device for detecting NAD(P)H is obtained. From probes with a known concentration of NADH and by measuring the reaction in percent at 660 nm, the curve according to Figure 5 is obtained. This curve can serve as a calibration curve for determining unknown NADH concentrations.

Methods for the formation of NAD(P)H from NAD(P)-dependent dehydrogenase, corresponding substrate and NAD(P) are known. After corresponding preliminary reactions, the NADH test device can therefore be used for the detection of dehydrogenase substrates or for the detection of dehydrogenases themselves.

Example 5

Test device for the determination of ethanol using alcohol dehydrogenase and diaphorase. as well as 18-molybdenum diphosphate

a) Manufacture of the test device

A test device according to figure 6 is produced.

This test device is structured in such a way that the indicator system consists of a homogeneous suspension of

dripped onto absorbent paper and then dried for 30 min at 40 °C.

The enzyme is placed on a separate paper. The paper is moistened with a solution of and then dried at 40 °C for 20 min. The thus produced indicator paper (13) and enzyme paper (12) are optionally divided into 14 x 6 mm strips.

On a 350 pm thick, 9.8 cm long and 0.6 cm wide polyester foil, a 16 mm long, 6 mm wide and 0.25 mm thick piece of glass fiber wool with a surface weight of 25 g/m<2> is attached as a transport body ( 14), a 6 mm wide, 6 mm long and 700 pm thick glass fiber wool piece with a basis weight of 60 g/m<2> as the erythrocyte secretion body (15) as well as a protective net (16) of "Scrynel PE280HC red" with the targets 6 x 8 mm and a mesh width of 280 pm, using a strip of tape (18) as shown in figure 6. Enzyme paper (12) and indicator paper (13) are fixed together with a 200 pm thick, 15 mm long and 6 mm wide, transparent polycarbonate foil (11) on the carrier foil (17) using a drop of glue (19), in such a way that (11), (12) and (13) do not come into contact with each other, but are brought into contact as well with each other as in liquid contact with a liquid located in the transport body (14), by means of pressure.

b) Determination of ethanol

On the protective net (16) in the test device according to

figure 6, prepared according to a), 32 pl of blood is applied. After 60 seconds, using pressure on the transparent foil (11), the enzyme paper (12) and the indicator paper (13) are pressed against the transport body (14) containing serum. After 120 seconds, the remission is measured in percent at 642 nm through the transparent foil (11). By adding known but different ethanol concentrations in blood, a curve according to Figure 7 is obtained. This curve can serve as a calibration curve for determining an unknown ethanol concentration in a probe.

Example 6

Determination of β-galactosidase (EC 3.2.1.23)

The following solutions were applied:

Buffer: 0.1 M HEPES, 20 mM magnesium chloride, pH 6.8 Substrate: 50 mM p-aminophenyl-p-galactoside (prepared from p-nitrophenyl-p-D-galactoside by hydrogenation with hydrogen on Pd-charcoal according to "Organikum" (VEB Deutscher Verlag der

Wissenschaften, 15th edition, Berlin 1976) in water Neutral-

sator: 1 N caustic soda

Indicator: 100 mg/ml 18-molybdenum diphosphoric acid and 50 mg/ml

phenyl-trimethyl-ammonium chloride in water

Enzyme: 180 U/ml in buffer (the stated enzyme activity refers to the use of o-nitrophenolgalactoside as substrate)

In a test reaction mixture was used:

After incubation for 4.5 min, the reaction mixture was centrifuged for 1/2 min, the supernatant was discarded, the precipitate was dissolved in 1 ml of dimethyl sulfoxide, and the extinction (E) was immediately measured. The results obtained are shown in table 3.

The resulting calibration curve from these values can be used to determine β-galactosidase content in unknown probes.

Using 50 mM p-aminophenyl phosphate (prepared according to L.H. De Riemer, CF. Meares, Biochemistry 20, 1606

(1981), as substrate in the recipe according to example 6, and by using 1 M diethanolamine, 1 mM magnesium chloride, 0.1 mM zinc chloride, pH 9.8, as buffer, determination of the enzyme alkaline phosphatase can be carried out. With known enzyme concentrations, as in example 6, a linear relationship between enzyme concentration and extinction at 800 nm was found.

Example 8

Test device for the detection of γ-glutamyl transferase

(EC 2.3.2.2)

Analogous to example 1, a test device was prepared, however with the following changes: In the second reagent layer, the buffer (2 wt% "Keltrol F" in 0.2 M citrate buffer, pH 6.0) was replaced with 2 wt% "Keltrol F " in water, N,N-bis-(2-hydroxyethyl )-p-nitroso-aniline x HCl and glucose oxidase were omitted. Between the adhesive layer (2) and the second reagent layer (3) was inserted a paper that had been soaked with substrate, prepared as follows: A tea bag paper (12 g/m<2>) was soaked with a solution of 250 mM glycylglycine and 20 mM γ-L-glutamyl-3-carboxyl-1,4-phenylenediamine (prepared according to EP-A-0,103,823) in 250 mM Tris buffer, pH 7.6, and dried for 20 min at 50°C.

In 0.1 M tris buffer containing 10 mg/ml bovine serum albumin, pH 7.5, a dilution series of γ-glutamyltransferase was prepared. 10 ul of this solution was applied to a test device described above. The remission values in percent were measured at a wavelength of 950 nm after 1 min at 37 °C, and these values are listed in table 4.

The thus obtained curve can be used to determine unknown γ-glutamyltransferase contents in liquids.

Example 9

Test device for the detection of acid phosphatase (EC 3.1.3.2)

Analogously to Example 8, a test device for the detection of acid phosphatase was obtained when the tea bag paper was soaked with 10 mM p-aminophenyl phosphate (prepared according to L.H. De Riemer, CF. Meares, Biochemistry 20, 1606, 1981), in 0.1 M citrate buffer, pH 5.0.

Claims (12)

1. Use of a sparingly soluble salt of a heteropolyacid for the determination of an analyte, in which the analyte is an electron-rich, aromatic amine, or in which the analyte in a preliminary reaction with one or more further compounds or enzymes lead to such an electron-rich, aromatic amine. 2. Method for determining an analyte by means of heteropoly blue formation, characterized in that the analyte is an electron-rich, aromatic amine or that the analyte is reacted with a compound, or one enzyme, which leads to the formation of an aromatic, electron-rich amine which is brought into contact with a sparingly soluble salt of a heteropolyacid. 3. Method according to claim 2, characterized in that a sparingly soluble salt of a heteropolyacid of molybdenum, molybdenum and tungsten, vanadium and molybdenum or vanadium and molybdenum and tungsten is used, with phosphorus, arsenic, silicon or germanium as heteroatom. 4. Method according to one of claims 2 or 3, characterized in that a salt in which the cation is larger than the ammonium ion is used as a sparingly soluble salt of a heteropolyacid. 5. Method according to one of the claims 2-4, characterized in that the sparingly soluble salt of a heteropolyacid is such a salt where the cation has general formula I in which R<1>, R<2>, R<3> and R<4> are the same or different and denote one alkyl, aryl or aralkyl residue, or hydrogen, provided that not all residues are equal, or whereby two residues together form an alkylene residue, and X denotes a phosphorus atom or nitrogen atom. 6. Method according to one of claims 2-4, characterized in that a salt with a cation from the group of quaternary nitrogen heteroaromatics is used as a sparingly soluble salt of a heteropolyacid. 7. Means for the determination of an electron-rich, aromatic amine or another analyte by means of heteropoly blue formation, characterized in that for the determination of an electron-rich, aromatic amine it contains a sparingly soluble salt of a heteropolyacid, or the compounds necessary for this purpose, or that for the determination of another analyte it contains a compound, or an enzyme, which contains a compound which, together with the analyte, leads to the formation of an electron-rich, aromatic amine and a sparingly soluble salt of a heteropolyacid, or the compounds necessary for this purpose. 8. Means according to claim 7, characterized in that it contains a sparingly soluble salt of a heteropolyacid of molybdenum, molybdenum and tungsten, vanadium and molybdenum or vanadium and molybdenum and tungsten, with phosphorus, arsenic, silicon or germanium as heteroatom. 9. Agent according to one of claims 7 or 8, characterized in that it contains, as a sparingly soluble salt of a heteropolyacid, such a salt whose cation is larger than the ammonium ion. 10. Agent according to one of claims 7-9, characterized in that it contains, as sparingly soluble salt of a heteropolyacid, a salt where the cation has general formula I in which R<1>, R<2>, R3 and R4 are the same or different and denote one alkyl, aryl or aralkyl residue, or hydrogen, with the proviso that not all residues are the same, or whereby two residues together form an alkylene residue, and X denotes a phosphorus atom or nitrogen atom. 11. Agent according to one of claims 7-9, characterized in that it contains, as a sparingly soluble salt of a heteropolyacid, such a salt with a cation from the group of quaternary nitrogen heteroaromatics. 12. Use of a sparingly soluble salt of a heteropolyacid for the preparation of a means for determining an analyte by means of heteropolyblue formation.